GATA-3 regulates the self-renewal of long-term hematopoietic stem cells

Abstract

The transcription factor GATA-3 is expressed and required for differentiation and function throughout the T lymphocyte lineage. Despite evidence it may also be expressed in multipotent hematopoietic stem cells (HSCs), any role for GATA-3 in these cells has remained unclear. Here we found GATA-3 was in the cytoplasm in quiescent long-term stem cells from steady-state bone marrow but relocated to the nucleus when HSCs cycled. Relocation depended on signaling via the mitogen-activated protein kinase p38 and was associated with a diminished capacity for long-term reconstitution after transfer into irradiated mice. Deletion of Gata3 enhanced the repopulating capacity and augmented the self-renewal of long-term HSCs in cell-autonomous fashion without affecting the cell cycle. Our observations position GATA-3 as a regulator of the balance between self-renewal and differentiation in HSCs that acts downstream of the p38 signaling pathway.

Figure 1. Gata3 is actively transcribed in LT- but not IT-HSC

(a) GFP fluorescence intensity distributions in bone marrow cells from wild-type or Gata3^GFP/+ mice expressing a GFP cassette under control of the endogenous Gata3 promoter. Total bone marrow cells were gated on forward and side scattering channels to exclude outliers. (b) Erythroid reconstitution from C57BL/6J-GPI1b-Gata3^+/+ (left) or C57BL/6J-GPI1b-Gata3^GFP/+ (right) bone marrow cells fractionated according to eGFP fluorescence and transferred in competition with wild-type cells of host genotype into lethally irradiated C57BL/6J-GPI1a mice. Blood samples were analysed at 8 and 32 wk post-transplant (3 mice per fraction in each of 2 independent experiments). Results from both experiments were pooled. The number of 8 wk (intermediate-term, top) and 32 wk (long-term, bottom) erythroid repopulating cells per fraction was calculated. The results for each fraction were normalized to percentage of total repopulating cells recovered from all 3 fractions. The number of intermediate term cells was the number calculated in a fraction at 8 wk minus the number determined at 32 wk.

Figure 2. GATA3 protein is expressed in LT- but not IT-HSC

(a) Immonufluorescence analysis of GATA3 expression in LSKRα₂^lo and α₂^hi cells. LSKRα₂^lo and α₂^hi cells were sorted, fixed, permeabilized and stained for GATA3. Representative images are shown. GATA3 staining was detected in approximately 1/3 of LSKRα₂^lo but not in α₂^hi cells. (b, e) Quantification of GATA3 flurorescence intensity in purified HSC. Total fluorescence signal was measured in individual cell images stained with mouse anti-GATA3 (left panel) or isotype control (IgG, right panel). Frequencies were normalized to a mode of 100 for α₂^hi cell images and equal total areas on each plot. The proportion of cells expressing GATA3 above background was 30% and 85% for LSKRα₂^lo and LSKRα₂^loSLAMF1^hi cells respectively. Results were aggregated from 5 and 3 independent separations respectively. (c) Isolation of LSKR α₂^loSLAMF1^hi and α₂^hiSLAMF1^lo cells. Events shown were gated on LSKR parameters. (d) High functional purity of the LSKRα₂^loSLAMF1^hi cell fraction. 50 sorted cells were coinjected with 10⁶ bone marrow cell competitors of host genotype into lethally irradiated recipients in 2 independent experiments. Historically this competitor dose would contain about 50 LT-HSC . Donor red blood cell reconstitution was measured at 8 wk intervals after transplantation with SEMs indicated. Achievement of 50% donor reconstitution at 24 – 32 wk is indicative of functional homogeneity of the LSKRα₂^loSLAMF1^hi fraction.

Figure 3. GATA3 relocalizes to the nucleus in cycling cells and the effect is inhibited by p38α inhibitors

(a) Subcellular localisation of GATA3 in quiescent and cultured LT-HSC. LSKRα₂^lo cells were stained for GATA3 and DNA counterstained with DAPI, directly after sorting or after culture. (b,c) Whole cell and nuclear fluorescence pixel intensities were summed for individual cells in microscopy images. The nuclear perimeter was traced in the DAPI-stained images and duplicated in the GATA3-stained images. The measurements are plotted as the mean ratio of summed nuclear to cytoplasmic fluorescence pixel intensities (n=6 or 7 cells for each value) with SEMs and 1-tail T test probabilities indicated (* = .031; ** = .00026). (d) Phospho-p38 MAPK staining in cultured LT-HSC. LSKRα₂^loSLAMF1^hi cells were cultured for 1 h in serum- and cytokine-free medium after which serum and cytokines were added. Controls received medium without serum and cytokines. After a further 15 min incubation cells were stained with anti-phospho-p38α antibody. Pixel intensities were summed for individual cells in fluorescence microscopy images. Frequencies were normalized as in figure 2b. Results are aggregated from 3 independent experiments. (e) Effect of p38α inhibitors. LSKRα₂^loSLAMF1^hi cells were cultured for 2 d with cytokines and SB239063 prior to staining and assessment of GATA3 localization by confocal microscopy. (f) Quantitation of GATA3 fluorescence localization in the presence of p38α inhibitors. Means + SEM from 2 independent experiments are shown with 1-tail T test probabilities (* = .026; ** = .00090, *** = 4.4E-04).

Figure 4. Poly(I:C) treatment induces HSC cycling and GATA3 relocalization in vivo and reduces the long-term reconstituting capacity of LSKRα₂^lo cells

(a) Cell cycle analysis in LSKα₂^loSLAMF1^hi cells at 1 and 10 d after in vivo treatment with poly(I:C). Cells were stained with anti-human Ki67 and Hoechst 33342 and analyzed by flow cytometry (left). Cycle phase distributions are plotted (right) showing means + SEM of 2 independent experiments. (b) Subcellular localization of GATA3 in LSKRα₂^lo cells after poly(I:C) treatment analysed by confocal microscopy. Whole cell and nuclear fluorescence intensities were summed in images of individual GATA3-positive cells (n=6 for each condition). The ratios of nuclear to cytoplasmic intensities are plotted (right) showing means + SEM. UT = Untreated. (c) 100 LSKRα₂^lo cells from control or poly(I:C) treated mice were injected in competition with 10⁶ bone marrow cells of host genotype into lethally irradiated recipients and erythroid reconstitution was tracked. Means and SEMs from 5 independent experiments are plotted.

Figure 5. Gata3 excision has little effect on steady state bone marrow and blood populations

Gata3^fl/f (“Control”) or Gata3^fl/fl-Mx1-Cre (“Excised”) mice were treated with poly(I:C) and analyzed 3 – 9 months later (“steady state”). (a) Myeloid and lymphoid cells (proportion of total cells, n=7 independent experiments) in blood and absolute numbers of phenotypic LT- and IT-HSC (n=4 and 3) in steady state bone marrow. (b) Cell cycle analysis in LSKα₂^loSLAMF1^hi bone marrow cells in steady state bone marrow or 1 d after a new poly(I:C) treatment. The bar graphs show means + SEM of 2 independent experiments. (c) Representative PCR reactions on DNA for assessment of Gata3 excision at the indicated times after treatment with poly(I:C). Image contrast and gamma were adjusted to allow visualization of the faint Gata3^fl band. 1, bone marrow 2 wk; 2, bone marrow 8 wk; 3, bone marrow 24 wk; 4, blood myeloid 7 mo; 5, blood myeloid 10 mo; 6–8, LSKR clones 10 d after excision; 9, Gata3^fl/fl unexcised control.

Figure 6. Regenerative activity and self-renewal are enhanced in vivo and in vitro after Gata3 deletion

(a) Gata3^fl/fl-Mx1-Cre (“excised”) and control Gata3^fl/fl (“intact”) mice were treated with poly(I:C). 10⁶ marrow cells taken 10 d later were injected with 10⁶ host-genotype bone marrow cells into irradiated recipients. Donor red cells were measured at intervals post-transplant (left). Proportions of donor myeloid and lymphoid cells in blood were measured at 32 wk (right). Means and SEMs of 3 independent experiments are indicated. (b) Gata3-deleted bone marrow cells were transplanted into irradiated 1° recipients with normal competitors. After 24 wk, bone marrow was transfered to irradiated 2° recipients. The bar graphs show the cumulative fold-expansion in LT-HSC numbers in 1° and 2° recipients with SEMs and 1-tail T test probabilities indicated. (c) Gata3^fl/fl or Gata3^fl/fl-Mx1-Cre^l bone marrow cells were injected with normal competitors into irradiated wild type recipients. Hosts were treated with poly(I:C) 8 wk later. At 24 wk, bone marrow cells were assayed competitively in vivo for long-term erythroid reconstituting activity. The bar graph shows the fold-expansion in LT-HSC numbers calculated to have occurred in the 1° recipients with SEMs indicated (single experiment, 3 – 5 mice per point, * = .04, 1-tail T test). (d) Bone marrow from Gata3^fl/fl-Mx1-Cre^l and control Gata3^fl/fl mice was analyzed 2–3 months after treatment with poly(I:C). Single LSKRα₂^lo (n=3 independent experiments) or LSKRα₂^loSLAMF1^hi (n=1) cells were cultured per well with serum and cytokines, and cell numbers were recorded every 4–8 hr. Each point represents the sum of cells in 30 wells. (e) LSKRα₂^loSLAMF1^hi cells, 25, from Gata3^fl/fl-Mx1-Cre or control Gata3^fl/fl mice treated 3 – 5 months earlier with poly(I:C) were cultured at 1 per well, and also assayed competitively in vivo for long-term erythroid reconstitution. After 7 d all harvested cells were again assayed in vivo. The bar graph shows SEM and mean relative number of LT-HSC recovered from 7 d cultures (“Out”) relative to the number initiating the cultures (“In”) in 2 independent experiments.